The production of high value therapeutic or diagnostic proteins by eukaryotic cells in the pharmaceutical or commercial manufacturing environment is subject to stringent constraints with regard to available fermentation vessel volume and fermentation process time. From the perspective of a manufacturing fermentation suite over a period of time, production efficiency comes from maximizing the rate of protein production per unit time while in operation, and minimizing the operational down time, and minimizing the fraction of operating time during which the production rate is low.
One broad approach to increasing protein productivity in the manufacturing setting is to increase the density of the cells in the vessel. Considerable increases in cell density can be achieved through the development of appropriately balanced culture medium formulations, by nutrient feeding approaches, and by perfusion of medium through systems that retain the cells. These approaches, in addition to increasing cell density, can also be effective in increasing the overall lifetime of batch cultures.
Another basic approach to increasing manufacturing productivity is to increase the rate of protein production by the cells, themselves, i.e., increasing the specific cellular productivity (amount of protein produced/cell/day). Traditionally, the way in which cells with high rates of productivity are attained comes about as a multi-step process in which, first, a large population of cells are transected with the gene for the protein of interest, and second, single cell clones of the larger population are grown out, and the higher producing cell lines are identified and selected for further cell line development. The heterogeneity with regard to protein production rate that manifests in a transfected cell population is generally ascribed to differences primarily in the position of integration of the new genetic material into the host cell's genome and secondarily in the total number of DNA sequences integrated (the copy number). Accordingly, methods for targeting the site of heterologous DNA integration into the host genome have also been developed, such that the DNA sequences can be integrated into sites associated with high levels of transcription into RNA. Examples of these approaches are provided by U.S. Pat. Nos. 5,648,267, 5,733,779, 6,017,733 and 6,159,730 pertaining to the NEOSPLA vector, and U.S. Pat. Nos. 5,830,698 and 5,998,144 pertaining to homologous recombination.
Also, co-transfection of the gene for the protein of interest with another gene for a protein that confers resistance to toxic agent offers a way to select for high producing cells. By exposing growing cells to increasing concentrations of such a toxic agent, cells with higher copy number of the resistance gene are selected. These cells are often found to have undergone a process of genomic amplification of the resistance gene. Co-amplification of the adjacent gene coding for the protein of interest often leads to an increase in the level of cellular specific production of the desired protein (Ringold, J. Mol. Appl. Genetic 1(3): 165-75 (1981); Kaufman, J. Mol. Biol. 159(4): 601-21 1982).
In an area of biological research with an origin entirely separate from the biochemical engineering of cell culture manufacturing processes, a great deal has been learned over the past decade about apoptosis, a process of programmed cell death. The conceptual breakthrough accompanying these developments is that there is a natural physiological process, which results in cell death. Cell death, of course, can also occur as a direct and immediate result of trauma, which destroys cell integrity, whereupon the cell or its remnants proceed to necrose. Apoptosis can be initiated by a wide variety of circumstances or stimuli, natural or developmental in nature, as part of a disease process, or in response to physiological stress. Developmental processes, whereby organs mature or reorganize, for example, can involve the death of specific kinds of cells, to make way for the emergence of other new kinds of cells. Understanding apoptosis at the genetic and biochemical levels, and the search for ways in which to intervene, either to promote it or to stop it, has thus emerged as an area of considerable interest in medical sciences.
Apoptosis, however, has also become appreciated as a major route for the cell death that occurs as a matter of course during in vitro culture of mammalian cells. A basic form of cell culture, done both in the laboratory and in large scale manufacturing environments, is termed batch culture. A batch culture is initiated with a healthy, actively growing cell inoculant; the culture grows to a peak cell density, enters a period of decline, and runs a course until the cell population dies off. The latter declining phase of cell culture is a time when the environment becomes stressful to the cells, as nutrients become depleted, and the concentrations of toxic metabolites increases. Additionally, the fermentation environment, itself, can offer other stressful factors, such as shear forces created by the hardware, fluid and gas turbulence associated with mixing, and the movement of the culture through pipes and lines. All of these factors (nutrient limitation, toxic agents in the medium, and physical stresses) have been demonstrated to initiate apoptosis in cells in culture, and thus to play a role in the limiting the life expectancy of the culture as a whole.
Several of proteins that play key roles in the apoptotic process have recently been discovered, and their genes isolated and cloned. E1B-19K is an adenovirus protein, which is an anti-apoptotic member of the Bcl-2 protein family. The cell's response to an infection is to initiate the apoptosis cascade. This defense mechanism is used to eliminate infected cells from tissues. However, certain viruses have developed a method to combat the apoptosis response by encoding proteins expressed early in the infection process that suppress apoptosis. During adenoviral infection, the viral gene expression in cells is regulated by the E1 region of the adenovirus genome. The E1 region is composed of two transcription units, E1A and E1B. The E1B unit encodes two distinct tumor antigens of adenovirus, 19 kDa and 55 kDa proteins. An inhibitor of apoptosis, E1B-19K's action is understood to be similar to that of anti-apoptotic protein Bcl-2, which inhibits apoptosis at multiple stages in the cell death pathway. One mechanism of apoptotic suppression is thought to be through stabilization of the mitochondrial membrane, thereby preventing release of cytochrome C and other pro-apoptotic factors in the cytosol. (Vander Heiden Cell 91 (5): 627-37 (1997)). Cytochrome C is involved in binding to Apaf-1 in a complex involved in the initiation of the caspase cascade. Zou J. Biol. Chem. 274 (17): 11549-56 (1999). Bcl-2 family members (Bcl-XL) also block activation of Apaf-1 by directly binding to Apaf-1. (Hu et al., J. Biol. Chem 273 (10): 5481-5 (1998); PNAS 95 (8): 4386-91 (1998)).
Another anti-apoptotic protein is Aven, a ubiquitous membrane protein. Aven's anti-apoptotic activity is attributed to its ability to inhibit caspase 9 activation by interfering with the self-association of the caspase activator Apaf-1 (Chau Mol. Cell. 6:31-40 (2000)). Aven has also been shown to enhance the anti-apoptotic activity of Bcl-XL in BHK (baby hamster kidney) cells, which suggests an interaction between Aven and Bcl-XL (Chau (2000)).
All of the above described various approaches to increasing protein productivity have succeeded in practice to varying degree. Nevertheless there is still a need to develop methods to increase production of cell-related product, e.g., recombinant proteins, especially in large-scale commercial production. In addition, there is a need to develop methods for prevention or delaying programmed cell death.